Pulmonary endothelial cells (EC) function as a complex tissue with the ability to respond to external stimuli by regulating fluid flow, trafficking inflammatory cells into the lung parenchyma or airways, creating new blood vessels, and undergoing apoptotic death. EC are continuously exposed to shear stress and cyclic stretch, mechanical forces dramatically enhanced in the presence of acute lung injury and mechanical ventilation. The Project Leader has shown each of these key EC processes to involve the critical participation of the myosin light chain kinase (MLCK)-driven actin cytoskeleton. MLCK phosphorylates MLCs on Ser19 and Thr18 producing spatially-localized tension through actomyosin contraction. Studies from the Garcia laboratory first revealed that EC express a unique high molecular weight MLCK isoform. In our initial PPG (1998-2003), we cloned EC MLCK to understand its structure and function. We identified four alternatively splice variants, which yield dramatic splice-specific cellular function. For example, EC MLCK1 but not EC MLCK2, is a substrate for p60src and when Tyr phosphorylated, exhibits unique upregulation of enzymatic activity. Furthermore, Tyr phosphorylation of EC MLCK1 enhances its stable association with the actin-linking protein and p60src substrate, cortactin. We have shown cortactin to localize with EC MLCK in cortical structures such as lamellipodia, and to participate in EC cytoskeletal rearrangement. Additional strategies (yeast 2 hybrid assay) yielded the clinically-relevant cytokine macrophage migration inhibitory factor as an EC MLCK binding partner, providing for potentially exciting pathophysiologic regulation, particularly in the context of acute lung injury. Finally, we have shown EC MLCK to be a critical participant in TNF-induced EC apoptosis with compelling data suggesting EC MLCK as a target for caspase cleavage. To investigate these key MLCK interactions, SA #1 will explore p60src-EC MLCK interaction in cytoskeletal rearrangements in human lung endothelium subjected to biophysical mechanical forces. SA #2 will characterize cortactin-EC MLCK interaction in lung endothelial cytoskeletal rearrangement. SA #3 will define EC MLCK-macrophage migration inhibitory factor (MIF) interaction and its role in human lung endothelial pathobiology. SA #4 will define the role of EC MLCK in both the initiation and execution of lung EC apoptosis.